Effects of Exposure to a Model Dioxin-like Compound on the Transcriptome of White Sturgeon (Acipenser transmontanus)
Jon Doering1,2, Steve Wiseman1 , Shawn Beitel1,2, Sarah Patterson1,2, John P. Giesy2,3,4, Markus Hecker2,5
1.Toxicology Graduate Program, University of Saskatchewan, Saskatoon, SK , CA 2.Toxicology Centre, University of Saskatchewan, Saskatoon, SK, CA 3. Dept. Veterinary Biomedical Sciences, University of Saskatchewan,
Saskatoon, SK, CA 4. Dept. of Biology & Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China 5. School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, CA
Background
Results and Discussion
• Worldwide many species of sturgeons (Acipenseridae) have faced
massive population declines with some species nearing extinction.
• These declines are attributed to a variety of human activities including
pollution, with dioxin-like compounds representing one contaminant of
concern.
Table 1: Xenobiotic Responses
Transcript
CYP1A
CYP2K1
CYP3A
Phase II
GST
UDP
Sulfotransferase
Phase III
Multi-drug
Resistant Proteins
• The pleiotropic responses of exposure to dioxin-like compounds are
mediated through the aryl hydrocarbon receptor (AhR) signaling pathway.
• However, little is known about the transcriptional responses of AhR
activation in sturgeons or the resulting toxic responses.
Juvenile
White Sturgeon
Fold-change
Phase I
Up-regulated
22%
Figure 1:
Down-regulated
78%
Figure 3: Global Gene Expression Following
Exposure to Beta-Naphthoflavone
Up-regulated
1) How responsive are sturgeon to dioxin-like compounds?
2) Which biological processes are affected?
3) What are possible toxic responses in sturgeons?
Methods
Exposure Experiment: White sturgeon (Acipenser transmontanus) were
injected intraperitoneally (I.P.) with one of two doses of a model dioxin-like
compound, β-naphthoflavone (βNF) dissolved in corn oil at 0 or 50mg/kg-bw.
Liver and gill samples were collected three days following injection.
Biological Process
Metabolic Processes
Xenobiotic Responses
Membrane Structure
Stress Response
Misc. Biological Processes
Other
up to 109x
• Greater than ¾ of the altered transcripts were
down-regulated (Figure 3).
Percentage
22%
15%
13%
7%
16%
27%
Biological Process
Membrane Structure
Transcription/Translation
Metabolic Processes
Immune Response
Misc. Biological Processes
Other
• Depletion of dissolved oxygen is a growing concern in areas of
anthropogenic activity, and exposure to dioxin-like compounds might impair
the ability of sturgeons to respond to hypoxia.
• An up-regulation in transcripts known to be upregulated by dioxin-like compounds in other
fishes, such as phase I, II, and II xenobiotic
metabolism enzymes, were up-regulated in white
sturgeon (Table 1).
• Additionally, exposure to dioxin-like compounds might indirectly cause
deformities in sturgeon embryos through altered expression of ARNT.
• Next-generation sequencing technologies, such as Illumina, could prove
useful in the discovery of novel biological responses to contaminants in nonmodel species through the adverse outcome pathway framework (Figure 5).
• Consistent with previously published work on
white sturgeon, the classical biomarker of
exposure to dioxin-like compounds, CYP1A, was
up-regulated (Table 1; Doering et al, 2012).
Down-regulated
Down-regulated
used for whole transcriptome analysis
of white sturgeon liver.
Illumina and RNA-Seq: Exposed and unexposed liver transciptomes were
analyzed by use of Illumina RNA-Seq after de novo assembly of a reference
transcriptome. Samples were sequenced on an Illumina HiSeq 2000 (100-bp
paired end reads). Sequencing was performed at the National Research Council
Plant Biotechnology Institution (Saskatoon, SK, CA). Contigs were de novo
assembled by use of the CLC Genomics Workbench 5.0. Identities of contigs
were determined by use of Blast2Go 2.5.0. Reads were mapped to the reference
transcriptome and abundance determined as RPKM. Transcripts were annotated
using gene ontology terms and Kyoto Encyclopedia of Genes and Genomes
mapping using Blast2Go 2.5.0.
13x
12x
24x
• Numerous critical pathways were altered which is consistent with other
studies with fish (Aluru & Vijayan, 2008).
Percentage
26%
22%
14%
3%
12%
23%
• Numerous biological processes
were affected by exposure to
beta-naphthoflavone in livers
of white sturgeon and were
consistent with findings in
rainbow trout (Aluru &
Vijayan, 2008).
• Inconsistent with other studies,
only one reproductive gene,
extrogen receptor alpha, was
altered in white sturgeon livers.
Figure 5: Adverse outcome pathway framework
References
Aluru N & Vijayan M. (2008). Aquat. Toxicol. 87. 1-12.
• A lesser percentage of immune
responsive genes were altered
in white sturgeon livers
compared to other studies.
Doering J, Wiseman S, Beitel S, Tendler B, Giesy J, Hecker M. (2012). Aquat. Toxicol. 114-115, 125-133.\
Pohjanvirta R. (2012). John Wiley & Sons. New Jersey.
Acknowledgements
Figure 2: Illumina HiSeq instrument
RNA Extraction: Total RNA was extracted from livers of 0 mg/kg-bw and 50
mg/kg-bw exposed sturgeon. RNA from 3 individuals from each treatment group
were pooled before sequencing.
40x
88x
34x
• White sturgeon livers are highly responsive to exposure to a model dioxinlike compound, beta-naphthoflavone.
• Beta-naphthoflavone altered greater than 2,000
transcripts by greater than or equal to 2-fold in
livers of white sturgeon.
Table 3: Down-regulated Processes
Table 2: Up-regulated Processes
Therefore the objectives of this study were to identify:
Conclusion
Down-regulated
• It has long been known that the AhR shares the aryl hydrocarbon
receptor nuclear translocator (ARNT) with the hypoxia inducible factor
alpha (HIFa) and it has been speculated that exposure to dioxin-like
compounds might interfere with the hypoxia response through
competition for or depletion of available ARNT (Figure 4). However,
this phenomena not been observed in laboratory model fish species that
have been tested to date (Pohjanvirta, 2012).
4x
• However, inconsistent with other studies, a down-regulation in ARNT
and HIFa transcript with an up-regulation in AhR transcript was
observed in livers of white sturgeon following exposure to betanaphthoflavone (Figure 4).
2x
Up-regulated
5x
• It is currently unclear whether these responses will impact the hypoxia
response in sturgeons following co-exposure to dioxin-like compounds.
Figure 4: Crosstalk between Aryl Hydrocarbon Receptor and Hypoxia
pathways with transcript responses following exposure to beta-naphthoflavone.
• Additionally, ARNT is important in numerous developmental processes
and an altered expression of ARNT might impact proper development in
embryos of sturgeons exposed to dioxin-like compounds.
*
All experimental procedures were approved by the University Committee on
Animal Care and Supply (UCACS) at the University of Saskatchewan.
Thanks to the Kootenay Trout Hatchery for their donation of white sturgeon.